We propose to study characteristics and mechanism of transbilayer movement of phospholipid in the membrane of the Gram-positive bacterium, Bacillus megaterium. By pulse-radiolabeling, in conjunction with trinitrobenzenesulfonic acid (TNBS) labeling to distinguish phosphatidylethanolamine (PE) at the inner and outer faces of the membrane, it has been shown that PE is made and inserted at the inner face and then undergoes the rapid transbilayer movement. Thus an isotopic asymmetry of PE is generated across the membrane early in pulse-labeling. The decay of this asymmetry, reflecting transbilayer movement, can then be studied under a variety of conditions. In this way, the energetics of the movement, the influence of membrane physical properties (such as lipid phase segregation), and the possibility of specific catalysis by membrane protein, will be explored. To test further for involvement of catalytic protein, solubilization, fractionation, and reconstitution of membrane components will be undertaken. We will assay for reconstituted function by introducing isotopic asymmetry exogenously in the reconstituted liposomes (with the aid of phospholipid exchange proteins), and following its decay. The transbilayer movement must be important in membrane biogenesis, for distributing newly made lipid between the two faces of the membrane. It is likely that findings will apply to the eucaryotic endoplasmic reticulum also, where similar outlines of biogenesis apply. Use of TNBS has also indicated a compositional asymmetry of PE in the B. megaterium membrane, with one-third facing out and two-thirds facing in. With suitable impermeant probes, we will study the compositional distribution, and possible transbilayer movement, of other phospholipids, including phosphatidylglycerol and phosphatidylserine. We will also test the idea that differential interactions with ions allow maintenance of asymmetry despite the rapid transbilayer movement. These studies should elucidate the role of lipid asymmetry in membranes.